Cell electropermeabilization, that is to say cell permeabilization via the local delivery of electric pulses (EP), is increasingly used for the management and prevention of a wide range of human and animal pathologies, including cancer.
Cell membrane delimits two compartments, the cytoplasm and the extracellular medium, that present different ions concentrations thus creating a difference in the transmembrane potential. When an electric field is applied to the cells, it results on an induced transmembrane potential which superimposes the resting one (Mir et al., 2005). Above a threshold, a transient permeabilization occurs leading to an exchange of molecules between the cytoplasm and the external medium. This phenomenon consequent to the application of EP to the cells and leading to the loss of membrane permeability is called electropermeabilization. This technique has been used for three decades to enhance non permeant molecules uptake by cells.
Although the exact mechanism of electropermeabilization is still subject to debate, this technique paved the way for many biomedical applications, in particular for cancer treatments (Breton & Mir, 2011). One of them, called antitumor electrochemotherapy, consists in coupling EP directly applied to the tumor site with the administration of bleomycin or cisplatin, which do not spontaneously diffuse (or poorly) through the plasma membrane (Mir et al., 1991; Mir, 2006). Once entering the electropermeabilized cells, these two drugs generate DNA damages and trigger cell death. Not only drugs but also nucleic acids, which are non permeant molecules (Satkauskas et al., 2002; Andre & Mir, 2010), can be electrotransferred into cells using electropermeabilization. DNA has been successfully transferred into various tissues of living animals including skin, muscle, liver, tumor, cornea, lung, kidney, brain, bladder and testis (reviewed in Andre et al., 2008; Gothelf & Ghel, 2010). One promising use of the gene electrotransfer method concerns the field of DNA vaccination. Indeed, DNA vaccination has raised a great excitement since the early 90's. Wolff and collaborators first managed to transfer DNA into animal muscles. Once transfected, DNA molecules allowed the target cells to produce the encoded protein (Wolff et al., 1990). Tang et al. demonstrated that a protein encoded by a DNA transferred to skin cells by a biolistic method could trigger an immune response (Tang et al., 1992) and Barry et al. showed that gene vaccination with a plasmid encoding a pathogen protein protected the animals against a challenge with the relevant pathogen (Barry et al., 1995). This technology has been used for a wide range of applications from laboratory tools to licensed veterinary vaccines (Anderson et al., 1996) and is under development for the management of various acquired pathologies such as cancer, malaria, hepatitis B and C or for the prevention of some viral infections such as influenza or human immunodeficiency virus (clinicaltrials.gov) (Bergman et al., 2003).
Eventually, DNA vaccines possess manufacturing, accessibility and economic advantages compared to other vaccine technologies (Liu, 2011). Despite the fact that DNA vaccines offer a precise and flexible strategy for delivering antigens to immune cells and to mount a specific immune response, there were issues at the beginning for translating this technique from small rodents to larger animals and in fine to patients (Rochard et al., 2011). As a matter of fact, DNA vaccines were found to be weakly immunogenic partially due to the low cellular uptake of DNA molecules at the site of vaccination. This problem has been overcome by using gene electrotransfer which dramatically improves the performance of DNA vaccines (Li et al., 2012; Gothelf & Gehl, 2012).
However, in order to use electrotransfer as expected for vaccination strategies, the procedure has to be performed with a very specific procedure. Electrotransfer is a multistep process that relies on two different types of EP (Andre & Mir, 2010; Satkauskas et al., 2005; Favard et al., 2007). First, DNA has to be brought close to the target cells environment by injection at the expected site of vaccination (skin, muscle), then one or several short (about one hundred of microseconds) and intense (about one thousand volts per centimeter) pulses, called high voltage (HV) pulses, permeabilize reversibly the cell membrane. A defined lag time later, one or several long (about several hundred of milliseconds) and less intense (about one hundred volts per centimeter) pulses, called low voltage (LV) pulses are applied. LV pulses are meant to drive electrophoretically the DNA throughout the extracellular matrix all the way to the contact with the electropermeabilized membrane. At this point, no consensus exists regarding how DNA molecules cross the plasma membrane and get to the nucleus to be taken in charge by cell translational machinery (Escoffre et al., 2009).
Interestingly, no serious side effect was ever detected either in animals or in humans after DNA administration followed by electrotransfer (Fioretti et al., 2013). A study reported that after administration, a DNA vaccine was mostly located around the site of the injection, its local detection levels decreased rapidly over time, no gonadal tissue internalized it (very low risk of germ-line transmission) and the integration probability was very low since no viral protein was used (Dolter et al., 2011). Moreover, when DNA molecules are used for vaccination purposes along with the gene electrotransfer method, there is no pre- or post-treatment immunity issues contrary to viral vectors such as adenoviral vectors, thus allowing multiple administrations (homologous prime-boost DNA/DNA or heterologous prime-boost DNA/vector or DNA/protein) (Villemejane & Mir, 2009). Consequently, DNA vaccination combined with electrotransfer has gained interest in the last few years.
For what concerns the cancer pathology, DNA vaccines are meant to trigger an immune response against tumor-specific or tumor-associated antigens (Stevenson & Palucka, 2010). Indeed, cancer cells fool the immune system that cannot always efficiently initiate an immune response due to multiple complex mechanisms such as self-tolerance (Bei & Scardino, 2010), diverse immunosuppression mechanisms involving either regulatory T-cells or myeloid-derived suppressor dendritic cells (moDCs) (Lindau et al., 2013), molecules expressed at the surface of immune cells such as CTLA-4 (Kolar et al, 2009; Shevach, 2009) and PD-1/PD-1L interaction (Keir et al., 2008).
The ultimate goal of an efficient DNA vaccine delivered via the electrotransfer technology must be to generate the right kind of immune responses against the antigen encoded by the plasmid of interest. Although well described for intramuscular administration route (patent application WO 2007/026236) (Mir et al., 2005; Andre & Mir, 2010), very few is known about electrotransfer parameters in the skin, for vaccination purposes. The immune response should be intense enough and long lasting enough to generate positive therapeutic effects in patients with a specific pathology. Of note, the intensity of the immune response depends, at least partially, on the level of antigen expression (Lee et al., 1997; Kirman & Seder, 2003), which is itself closely correlated to the efficacy of gene transfer. Regarding gene electrotransfer efficiency in skin, it depends on several parameters including the intensity of EP and the type of electrodes used to deliver them (Gothelf & Gehl, 2010).